CN102731642A - Production technology of high-pure Apoa-I from fourth deposit of human blood plasma component - Google Patents
Production technology of high-pure Apoa-I from fourth deposit of human blood plasma component Download PDFInfo
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Abstract
The invention discloses a production technology of high-pure apolipoprotein Apoa-I from fourth deposit of a blood plasma component. The production technology is to prepare high-pure Apoa-I from fourth deposit of the blood plasma component through one step of centrifugation and one step of ion column chromatography. The production technology provided by the invention has advantages of simple equipment, convenient operation, few steps, short production period and high protein recovery rate, and is suitable for industrial large-scale production.
Description
Technical field
The invention belongs to biological pharmacy technical field, relate to a kind of production technique that from plasma component four depositions, prepares high purity aPoA poa-I.
Background technology
Lipophorin a-I (Apolipoprotein a-I is called for short Apoa-I) is that (High DensityLipoprotein, main lipophorin HDL) are the single polypeptide chain to RHDL, are made up of molecular weight 28.3kD 243 amino-acid residues.The HDL major function is to participate in SUV antiport (Reverse Cholesterol Transport; RCT); SUV in peripheral tissues's cell is shifted out and be transported to liver transform removing; Thereby have that (Atherosclerosis AS) takes place and the vital role of development, and the main undertaker of the Apoa-I anti-AS function that is HDL to atherosclerosis.Simultaneously, Apoa-I also has the antiendotoxic function of anti-inflammatory, is one of lipid metabolism research emphasis therefore.In addition, because Apoa-I has the liver target effect, in targeted drug research, good prospects for application is arranged also.
The preparation method commonly used of Apoa-I has ultracentrifugation, organic solvent precipitation method and high-efficient liquid phase technique etc. at present, though can obtain high purity Apoa-I, have some shortcomings clearly: 1. preparation amount is little, is inappropriate for industrial production; 2. the albumen yield is low, handles through multisteps such as ultra centrifugal, organic solvent deposit, column chromatographies, in the preparation process, has lost most of Apoa-I; 3. cost is high, needs expensive instruments such as ultracentrifuge; 4. poor stability, organic solvents such as ethanol, acetone, trichoroacetic acid(TCA) not only have physiology toxicity, and inflammable and explosive, unfavorable and safety in production.
On the other hand, plasma component four is human plasma remainders after the Cohn cold ethanol method is handled, and is dropped because of not utilizing, and its major protein composition is BSA and beta Lipoprotein.The present invention provides a kind of process method, and purifying obtains high purity Apoa-I from component four, and the blood plasma resource is utilized more fully.
Summary of the invention
The objective of the invention is to be precipitated as raw material with the human plasma component four that is not fully utilized as yet at present; Provide a kind of operation simple; Easy to operate, with short production cycle, yield is high; Be applicable to high-purity aPoA poa-I purifying process that large-scale industrialization is produced, product is used to treat cardiovascular and cerebrovascular diseases such as arteriosclerosis.
Technical scheme provided by the invention is centrifugally to add step ion column chromatography preparation high purity Apoa-I from plasma component four depositions through a step, thus have that equipment is simple, easy to operate, step is few, with short production cycle, protein recovery is high, be suitable for advantage such as large-scale industrialization production.
The present invention utilizes physicochemical property such as the iso-electric point of Apoa-I, and density etc. obtain suitable separation condition through acidity and the ionic strength of reconciling suspension-s; The albumen precipitation of Apoa-I is rich in centrifugal acquisition; Redissolve then and precipitate, last ion exchange column is selected suitable upward appearance and elution requirement for use; Realize the high efficiency separation of Apoa-I and foreign protein, reached the purifying purpose.
The key point of technical scheme of the present invention is that centrifugal acquisition Apoa-I albumen precipitation is then with a step anion-exchange chromatography purifying Apoa-I.In centrifugation step, obtain to be rich in the albumen precipitation of Apoa-I.In the anion-exchange chromatography step; Select suitable last column condition, Apoa-I is attached on the chromatography column with some foreign protein, and other foreign protein is then worn liquid with stream and removed; To contain certain density urea buffer solution wash-out, finally obtain high-purity Apoa-I protein solution then.
The Apoa-I technological process of production that the present invention proposes is referring to accompanying drawing 1
The concrete operations step is following:
(1) component four resolution of precipitates
Component four resolution of precipitates in damping fluid (about pH5.5～11.0), are stirred and make it abundant dissolving.
Said dissolving can be with damping fluid: acetate buffer, tris damping fluid or phosphate buffered saline buffer.It is 0～30 ℃ that the damping fluid temperature is used in dissolving, preferred 2-15 ℃.
Component four moist precipitates and lysate weight ratio generally were controlled at 1: 5～1: 20
(2) centrifugal acquisition Apoa-I deposition
In suspension-s, add certain density salt (0.2～6%W/W), reconcile pH value then to acid (pH4.8～6.8), cooling (3～20 ℃), high speed centrifugation then, collecting precipitation is Apoa-I and precipitates, and abandons supernatant.
Said salt can be sodium-chlor.
The pH regulator agent can be: aqueous hydrochloric acid, aqueous acetic acid etc.
High speed centrifugation is meant that centrifugal speed is more than 5000rpm.
(3) redissolution Apoa-I deposition
The Apoa-I deposition fully is dissolved in the damping fluid (about pH5.5～11.0), uses millipore filtration (like 0.45 μ m filter membrane) to filter then.
Said redissolution can be with damping fluid: acetate buffer, tris damping fluid or phosphate buffered saline buffer.The use damping fluid temperature of redissolving is 0～30 ℃, preferred 10.0-25.0 ℃.
The Apoa-I deposition generally was controlled at 1: 5～1: 20 with the lysate weight ratio
(4) viral inactivation treatment
Tween 80 and TNBP (tbp) that dissolving is good are added in the filtrating, are incubated at least 6 hours down at 25 ± 1.0 ℃, accomplish the first step viral inactivation treatment.
Preferable, the addition of tween 80 is a 1.0-1.1g/100ml filtrating; The addition of TNBP is a 0.3-0.32g/100ml filtrating.
(5) column chromatography
The Apoa-I that a last step is obtained filtrates with anion chromatography post on the suitable flow velocity, and Apoa-I and part foreign protein are attached on the chromatography column, and another part foreign protein stream is worn.With the buffer solution elution chromatography column that contains urea (2-8M), Apoa-I is then eluted, and through the SDS-PAGE electrophoresis detection, has obtained electrophoretically pure Apoa-I, and has not contained Apoa-I in the foreign protein elutriant basically then.
Need the pH value to be adjusted to 5.5～11.0 before the filtrating upper prop, ionic strength is adjusted to specific conductivity 0.5～15ms/cm.
The preferred DEAE FF of said anion chromatography post anion chromatography post.
The pH regulator agent can be selected from: aqueous hydrochloric acid or aqueous sodium hydroxide solution.
Ionic strength adjustor can be selected from: sodium-chlor, sodium-acetate etc.
Last appearance flow velocity is generally 60-240cm/h, preferred 100-200cm/h.
Wash-out can be selected from damping fluid: acetate buffer, tris damping fluid or phosphate buffered saline buffer.Wash-out uses the pH value of damping fluid to be 5.5-11.0.
Further, also can be through the Apoa-I of negatively charged ion column chromatographic isolation and purification solution through aftertreatment (like ultrafiltration etc.), inactivation of virus, sterile filtration, the canned liquid preparation of processing, or further process freeze-dried prepn after the freeze-drying.
Concrete, the aftertreatment of Apoa-I elutriant can for: the Apoa-I that elutes through ultrafiltration dialysis, is concentrated into suitable concn then, and adds stablizer.Adjust pH to 7.0 ± 02.
After concentrating, the preferred 6-8% of the concentration of Apoa-I in elutriant (g/100ml).
Stablizer can be selected from: common glycitols stablizer such as sucrose, and concentration is preferably 10-30wt%; Sorbyl alcohol or N.F,USP MANNITOL, preferred 5 ± 0.1% (g/100ml) of concentration; Also can be BSA, the preferred 0.5-2.5% of concentration (g/100ml)
Inactivation of virus can be above solution is filtered through nanometer film (like the DV20 nanometer film), accomplishes the second step viral inactivation treatment.
Characteristics of the present invention:
(1) this prepared Apoa-I has very high selectivity, and purity is very high, and liquid preparation or lyophilized powder purity can reach more than 95%, and the Apoa-I yield is also very high, can reach more than 70%.
(2) preparation process does not relate to organic solvent, easy-to-operate.
(3) pilot-scale experiment shows, the centrifugal purifying Apoa-I that combines with column chromatography is easy to realize the technology amplification, is fit to very much suitability for industrialized production.
Description of drawings
Fig. 1: human plasma aPoA poa-I technological process of production block diagram
Fig. 2: the SDS-PAGE electrophoresis result figure of continuous three batches of Apoa-I lab scale samples
Wherein 1,5,10 be respectively embodiment 1 Apoa-I resolution of precipitate liquid, 2,6,8 are respectively embodiment 1 urea elutriant initial section collects sample; 3,7,9 are respectively embodiment 1 urea elutriant main body collects sample; 4 is the lower molecular weight standard substance.
Fig. 3: the SDS-PAGE electrophoresis result figure of Apoa-I pilot scale freeze-drying sample redissolution liquid
Wherein 1 is lower molecular weight standard protein band, and 2 is the BSA band, and 3,4,5,6,7,8 is the protein band (Apoa-I) (different freeze-drying prescription) of freeze-drying sample redissolution liquid among the embodiment 4
Embodiment
Embodiment 1, the specimen preparation of Apoa-I lab scale
(1) component four resolution of precipitates and pre-treatment
Take by weighing component four deposition 50 grams (weight in wet base contains zeyssatite), be dissolved in the 450 gram acetate buffers (0～2 ℃), adjust pH is about about 6.0, fully stirs and makes it dissolving.
(2) centrifugal acquisition Apoa-I deposition
In suspension-s, add sodium-chlor its concentration is reached about 6%, reconcile then about pH value to 4.8, be cooled to about-3 ℃.Use Eppendorf centrifuge 5804R high speed centrifugation (9000rpm) then, collect the about 40g of deposition that is rich in Apoa-I, abandon supernatant.
(3) redissolution Apoa-I deposition
Apoa-I is precipitated 40g fully be dissolved in about about the 30 ℃ acetate buffer of 360g, use 0.45 μ m membrane filtration then.
(4) S/D handles
Add tween 80 and TNBP in filtrating, be incubated 6 hours down at 25 ℃.Accomplish the first step viral inactivation treatment.
Tween 80 dosage 1.0g/100ml filtrating, TNBP dosage 0.3g/100ml.
(5) column chromatography
The acidity pH of adjustment Apoa-I filtrating be about 6.50 with ionic strength be 13ms/cm; With (the DEAE anion chromatography column volume 30ml of DEAE anion chromatography post on the 120cm/h flow velocity; Chromatographic stuffing is DEAESepharose FF; Purifier apparatus is AKTA EXPLORER 100, and Apoa-I and some foreign protein are attached on the chromatography column, and part foreign protein stream is worn.With the acetate buffer wash-out DEAE chromatography column that contains urea (2mol/L), Apoa-I is at first eluted then; Collect the about 60ml of elutriant, with high density chlorination sodium solution washing DEAE chromatography column, most foreign protein wash-outs are removed again,, obtained electrophoretically pure Apoa-I, and do not contained Apoa-I in the foreign protein elutriant basically through the SDS-PAGE electrophoresis detection.
(6) Apoa-I elutriant aftertreatment
The Apoa-I that elutes dialyses with acetate buffer through millipore labscale TFF system ultrafiltration and concentration, be concentrated into about 7% then, and with N.F,USP MANNITOL to 5% as stablizer.Regulate about protein concentration to 5% and adjust pH to 7.0.
(7) nano-film filtration
Above solution through the DV20 nano-film filtration, is accomplished the second step viral inactivation treatment.
(8) sterile filtration and canned
Solution through nano-film filtration carries out sterile filtration again, presses the packing of 2ml/ bottle, becomes liquid preparation.
(9) or further process freeze-dried prepn through freeze-drying
Product detects through the SDS-PAGE method, and purity is more than 95%, and molecular weight is 28KD, and the cell that has Apoa-I through verification experimental verification combines activity, and the albumen that final affirmation obtains is Apoa-I albumen.Calculating the Apoa-I yield is 74.5% (weight ratio of Apoa-I in the Apoa-I of acquisition and the raw blood plasma component four).
(1) component four resolution of precipitates and pre-treatment
Take by weighing component four deposition 50 grams (weight in wet base contains zeyssatite), be dissolved in the 450 gram tris damping fluids (28-30 ℃), adjust pH is about about 10.0, fully stirs and makes it dissolving.
(2) centrifugal acquisition Apoa-I deposition
In suspension-s, add sodium-chlor its concentration is reached about 0.2%, reconcile then about pH value to 5.8, be cooled to about 20 ℃.Use Eppendorfcentrifuge 5804R high speed centrifugation (9000rpm) then, collect the about 40g of deposition that is rich in Apoa-I, abandon supernatant.
(3) redissolution Apoa-I deposition
Apoa-I is precipitated 40g fully be dissolved in about about the 5 ℃ tris damping fluid of 360g, use 0.45 μ m membrane filtration then.
(4) S/D handles
Add tween 80 and TNBP in filtrating, be incubated 6 hours down at 25 ℃.Accomplish the first step viral inactivation treatment.
(5) column chromatography
The acidity pH of adjustment Apoa-I filtrating be about 8.0 with ionic strength be 10ms/cm; With (the DEAE anion chromatography column volume 30ml of DEAE anion chromatography post on the 60cm/h flow velocity; Chromatographic stuffing is DEAESepharose FF; Purifier apparatus is AKTA EXPLORER 100, and Apoa-I and some foreign protein are attached on the chromatography column, and part foreign protein stream is worn.With the Tris buffer solution elution DEAE chromatography column that contains urea (4.5mol/L), Apoa-I is at first eluted then; Collect the about 60ml of elutriant, with high density chlorination sodium solution washing DEAE chromatography column, most foreign protein wash-outs are removed again,, obtained electrophoretically pure Apoa-I, and do not contained Apoa-I in the foreign protein elutriant basically through the SDS-PAGE electrophoresis detection.
(6) Apoa-I elutriant aftertreatment
The Apoa-I that elutes dialyses with the tris damping fluid through millipore labscale TFF system ultrafiltration and concentration, be concentrated into about 7% then, and with N.F,USP MANNITOL to 5% as stablizer.Regulate about protein concentration to 5% and adjust pH to 7.0.
(7) nano-film filtration
Above solution through the DV20 nano-film filtration, is accomplished the second step viral inactivation treatment.
(8) sterile filtration and canned
Solution through nano-film filtration carries out sterile filtration again, presses the packing of 2ml/ bottle, becomes liquid preparation.
(9) or further after freeze-drying, become freeze-dried prepn.
Product detects through the SDS-PAGE method, and purity is more than 95%, and molecular weight is 28KD, and the cell that has Apoa-I through verification experimental verification combines activity, and the albumen that final affirmation obtains is Apoa-I albumen.Calculating the Apoa-I yield is 76.8% (weight ratio of Apoa-I in the Apoa-I of acquisition and the raw blood plasma component four).
(1) component four resolution of precipitates and pre-treatment
Take by weighing component four deposition 50 grams (weight in wet base contains zeyssatite), be dissolved in the 450 gram phosphate buffered saline buffers (14-16 ℃), adjust pH is about about 8.5, fully stirs and makes it dissolving.
(2) centrifugal acquisition Apoa-I deposition
In suspension-s, add sodium-chlor its concentration is reached about 1%, reconcile then about pH value to 6.50, be cooled to about 0 ℃.Use Eppendorf centrifuge 5804R high speed centrifugation (9000rpm) then, collect the about 40g of deposition that is rich in Apoa-I, abandon supernatant.
(3) redissolution Apoa-I deposition
Apoa-I is precipitated 40g fully be dissolved in about about the 15 ℃ phosphate buffered saline buffer of 360g, use 0.45 μ m membrane filtration then.
(4) S/D handles
Add tween 80 and TNBP in filtrating, be incubated 6 hours down at 25 ℃.Accomplish the first step viral inactivation treatment.
(5) column chromatography
The acidity pH of adjustment Apoa-I filtrating be about 5.5 with ionic strength be 0.5ms/cm; With (the DEAE anion chromatography column volume 30ml of DEAE anion chromatography post on the 200cm/h flow velocity; Chromatographic stuffing is DEAESepharose FF; Purifier apparatus is AKTA EXPLORER 100, and Apoa-I and some foreign protein are attached on the chromatography column, and part foreign protein stream is worn.With the phosphate buffered saline buffer wash-out DEAE chromatography column that contains urea (6mol/L), Apoa-I is at first eluted then; Collect the about 60ml of elutriant, with high density chlorination sodium solution washing DEAE chromatography column, most foreign protein wash-outs are removed again,, obtained electrophoretically pure Apoa-I, and do not contained Apoa-I in the foreign protein elutriant basically through the SDS-PAGE electrophoresis detection.
(6) Apoa-I elutriant aftertreatment
The Apoa-I that elutes dialyses with phosphate buffered saline buffer through millipore labscale TFF system ultrafiltration and concentration, be concentrated into about 7% then, and with N.F,USP MANNITOL to 5% as stablizer.Regulate about protein concentration to 5% and adjust pH to 7.0.
(7) nano-film filtration
Above solution through the DV20 nano-film filtration, is accomplished the second step viral inactivation treatment.
(8) sterile filtration and canned
Solution through nano-film filtration carries out sterile filtration again, presses the packing of 2ml/ bottle, becomes liquid preparation.
(9) or further process freeze-dried prepn through freeze-drying
Product detects through the SDS-PAGE method, and purity is more than 95%, and molecular weight is 28KD, and the cell that has Apoa-I through verification experimental verification combines activity, and the albumen that final affirmation obtains is Apoa-I albumen.Calculating the Apoa-I yield is 73.2% (weight ratio of Apoa-I in the Apoa-I of acquisition and the raw blood plasma component four).
Embodiment 4, Apoa-I pilot scale specimen preparation
(1) component four resolution of precipitates and pre-treatment
Take by weighing 5 kilograms (weight in wet base contains zeyssatite) of component four deposition, be dissolved in 45 kilograms of phosphate buffered saline buffers (8～10 ℃), adjust pH is about about 7.5, fully stirs and makes it dissolving.
(2) centrifugal acquisition Apoa-I deposition
In suspension-s, add sodium-chlor its concentration is reached about 2%, reconcile then about pH value to 4.80, be cooled to about-1 ℃.Use Beckman whizzer (7000rpm) high speed centrifugation then, collect the about 4kg of Apoa-I deposition, abandon supernatant.
(3) redissolution Apoa-I deposition
Apoa-I is precipitated 4kg fully be dissolved in about about the 0 ℃ phosphate buffered saline buffer of 36kg, use 0.45 μ m membrane filtration then.
(4) S/D handles
Add tween 80 and TNBP in filtrating, be incubated 6 hours down at 25 ℃.Accomplish the first step viral inactivation treatment.
(5) column chromatography
The acidity pH of adjustment Apoa-I filtrating be about 11.0 with ionic strength be 15ms/cm; With DEAE anion chromatography post (3 liters of DEAE anion chromatography column volumes on the 120cm/h flow velocity; Chromatographic stuffing is DEAESepharose FF); Apoa-I and some foreign protein are attached on the chromatography column, and part foreign protein stream is worn.With the phosphate buffered saline buffer wash-out DEAE chromatography column that contains urea (8mol/L), Apoa-I is at first eluted then; Collect about 6 liters of elutriant, with high density chlorination sodium solution washing DEAE chromatography column, most foreign protein wash-outs are removed again,, obtained electrophoretically pure Apoa-I, and do not contained Apoa-I in the foreign protein elutriant basically through the SDS-PAGE electrophoresis detection.
(6) Apoa-I elutriant aftertreatment
The Apoa-I that elutes dialyses with phosphate buffered saline buffer through millipore pilot system ultrafiltration and concentration, is concentrated into then about 7%, with N.F,USP MANNITOL to 5%, regulates about protein concentration to 5% and adjust pH to 7.0.
(7) nano-film filtration
Above solution through the DV20 nano-film filtration, is accomplished the second step viral inactivation treatment.
(8) sterile filtration and canned
Filtrating through nano-film filtration is carried out sterile filtration again, presses the packing of 2ml/ bottle.
(9) freeze-drying
Through lyophilize, promptly process the Apoa-I freeze-dried prepn.
Product detects through the SDS-PAGE method, and purity is more than 95%, and molecular weight is 28KD, and the cell that has Apoa-I through verification experimental verification combines activity, and the albumen that final affirmation obtains is Apoa-I albumen.Calculating the Apoa-I yield is 72.1% (weight ratio of Apoa-I in the Apoa-I of acquisition and the raw blood plasma component four).
Claims (11)
1. a method of from human plasma component four depositions, separating Apoa-I comprises the following steps:
(1) with component four resolution of precipitates in the damping fluid of pH5.5～11.0;
(2) in suspension-s, adding weight percent is 0.2～6% salt, reconciles pH value to acid, be cooled to-3～20 ℃ and separate out deposition, and high speed centrifugation then, collecting precipitation obtains Apoa-I and precipitates, and abandons supernatant;
(3) redissolution Apoa-I deposition: the Apoa-I resolution of precipitate in the damping fluid of pH5.5～11.0, is used filtering with microporous membrane then;
(4) viral inactivation treatment;
(5) column chromatography: earlier will be on the Apoa-I filtrating that step (4) is handled the anion chromatography post, usefulness contains the buffer solution elution chromatography column of 2-8M urea and collects elutriant then, obtains electrophoretically pure Apoa-I solution.
(6) wash-out is collected liquid and carry out ultrafiltration, add stablizer, carry out sterile filtration behind the adjust pH, the back is canned.
2. the method for claim 1 is characterized in that, dissolving all is selected from acetate buffer, tris damping fluid or phosphate buffered saline buffer with redissolving in damping fluid and the step (3) with damping fluid in the step (1), and the temperature of damping fluid is 0-30 ℃.
3. the method for claim 1 is characterized in that, in the step (2), said salt is sodium-chlor.
4. the method for claim 1 is characterized in that, the viral inactivation treatment method of step (4) is: will dissolve good tween 80 and TNBP and be added in the filtrating of step (3) acquisition, 24-26 ℃ is incubated at least 6 hours down, accomplishes viral inactivation treatment.
5. method as claimed in claim 4 is characterized in that, the addition of said tween 80 is a 1.0-1.1g/100ml filtrating; The addition of said TNBP is a 0.30-0.32g/100ml filtrating.
6. the method for claim 1 is characterized in that, in the step (5), needs the pH value to be adjusted to 5.5-11.0 before the filtrating upper prop, and ionic strength is adjusted to specific conductivity 0.5-15ms/cm.
7. method as claimed in claim 6 is characterized in that, regulates pH and is selected from regulator: aqueous hydrochloric acid or aqueous sodium hydroxide solution, and regulate ionic strength and be selected from: sodium-chlor, sodium-acetate etc. with regulator.
8. the method for claim 1 is characterized in that, in the step (5), said anion chromatography post is a DEAE FF anion chromatography post, and last appearance flow velocity is 60-240cm/h.
9. like the described method of the arbitrary claim of claim 1-8, it is characterized in that in the step (5), wash-out is selected from damping fluid: acetate buffer, tris damping fluid or phosphate buffered saline buffer, wash-out use the pH value of damping fluid to be 5.5-11.0.
10. the method for claim 1 is characterized in that, the said stablizer of step (6) is glycitols stablizer or BSA.
11. the method for claim 1 is characterized in that, processes liquid preparation through the Apoa-I solution that step (6) obtains through sterile filling, or further processes freeze-dried prepn.
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| CN102731642B (en) | 2014-01-29 |
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